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Abstract

Background

Diabetic nephropathy (DN) is the major complication of diabetes; proteinuria is the
hall mark of DN. Currently, the treatment for proteinuria is mainly limited to angiotensin
converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs). According
to Traditional Chinese Medicine (TCM) theory, Chinese medicinals ‘securing essence
and tonifying the kidney’ may be appropriate for proteinuria. The most promising Chinese
medicinals and formulae are introduced in the present study to form a potent formula
for DN proteinuria. To make oral administration convenient, the formula will be processed
in the form of granules.

Methods/design

A randomized, multi-center pilot trial will be conducted. Forty eight participants
with DN will be randomly assigned to one of four treatment groups:

The following outcome measures will be used: the percentage change of the albumin-to-creatinine
ratio; and the changes in serum creatinine, glomerular filtration rate, fasting plasma
glucose and hemoglobulin from baseline to the end of the trial.

Discussion

It is notable that most published clinical trials which assessed the efficacy of TCM
on DN were of poor methodology and, therefore, their results have been invalidated.
It is necessary to carry out well-designed clinical trials to provide sound evidence.
The present trial is a study with potentially great value, for it will provide the
parameters for future randomized, placebo-controlled, clinical trials with large sample
sizes.

Keywords:

Background

The prevalence of diabetic nephropathy (DN) in China is not very clear; however, in
Beijing, the city which has the best medical service in China, the incidence of DN
in patients with type 2 diabetes is 35.7% and that of microalbuminuria is 13.6% [1].

In Traditional Chinese Medicine (TCM) theory, the kidney is a paramount organ. It
stores essence and the essence transforms Qi and produces blood. In addition, the kidney is the source of genuine Yin and genuine Yang. It is called ‘the prenatal base of life’ in TCM textbooks [2]. In the TCM system, there are many Chinese medicinals classified as tonifying and
replenishing medicinals. Many of them have the effects of replenishing Qi of the kidney or tonifying kidney Yang.

We have known that DN is the major complication of diabetes and proteinuria is the
hallmark of DN. DN is also defined by increased urinary albumin excretion (UAE) in
the absence of other renal diseases, which is categorized into stages according to
the level of UAE, that is, microalbuminuria (30 to 299 mg/24 hours) and macroalbuminuria
(300 mg/24 hours or greater) [3]. Currently, the treatment for proteinuria is largely limited to angiotensin converting
enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs) [4]. Although the clinical beneficial effects of many ACE inhibitors or ARBs have been
established [5], the efforts of looking for new drugs for DN that involve reducing proteinuria never
stop. For example, many researchers investigated astragalus for the treatment of DN
and the results seemed intriguing. Zhang et al. [6] systematically reviewed published animal experiments that had assessed the renal
protective effects of astragalus in diabetic rat models. Their results showed significant
beneficial effects of astragalus involving improved fasting blood glucose levels,
glomerular filtration rate (GFR), urinary albumin excretion rate, and thickness of
the glomerular basement membrane. Hong and co-workers [7] reported that astragalus might ‘play protective roles in diabetic nephropathy through
multiple pathways at the gene level’.

Is TCM really able to lower proteinuria in patients with DN? Current evidence has
tried to address this issue, but the picture remains unclear [8]. The present research looks at the DN-lowering proteinuria effect of TCM to answer
this question. To evaluate the effects of TCM accurately, the most promising Chinese
medicinals and formulae are used in the present study to form a potent formula for
the treatment of proteinuria of DN.

Because proteinuria, in TCM theory, results from the leakage of the essence of the
human body due to an insufficiency of the kidney, it is promising to look for Chinese
medicinals that secure the essence and tonify the kidney for the treatment of proteinuria.
A typical herb that secures essence and tonifies the kidney is used in the present
study, Flastem Milkvetch Seed. A famous classical formula securing the essence and
tonifying the kidney is also introduced in the present study for the treatment of
proteinuria (Water-land Two Elixirs). It consists of two Chinese medicinals, Gordon
Euryale Seed and Cherokee Rose Fruit. The first ingredient is grown in water and the
second one is grown on land, which is the reason why it is named ‘Water-land.’ This
formula is very effective for treating conditions such as spermatorrhea and enuresis
[9]. However, only securing essence is not enough; because Yin/Yang has been damaged, it is essential to repair the balance between them.

There is a therapeutic strategy in TCM theory, namely, using ‘cool’ and ‘warm’ herbs
together to balance Yin/Yang; this strategy is introduced in the present study. As the most conspicuous topic
in the area of TCM treatment for DN, astragalus may be a very potent Chinese medicinal
for proteinuria; therefore, it is also used in the present study. Both basic and clinical
studies have demonstrated that astragalus could exert beneficial effects on DN [10-12]. An in vitro study investigated the effect of calycosin and calycosin-7-O-β-D-glucoside. These
two major isoflavonoids in astragalus were tested with high glucose-induced rat mesangial
cell proliferation and advanced glycation end product (AGE)-induced human glomerular
endothelial cell apoptosis. The results suggested that both isoflavonoids had a significant
therapeutic potential to modulate the development and/or progression of DN [10]. Zhang and his co-workers studied the effects of astragalus polysaccharide (APS),
an aqueous extract from the astragalus membranaceus roots, on gene expressions of
nuclear factor-kappaB (NF-kappaB) and an inhibitory protein of NF-kappaB (IkappaB)
in experimental DN rats induced by streptozotocin. The results showed that APS improved
proteinuria and renal function and that the mRNA level of NF-kappaB in renal cortex
was decreased and IkappaB mRNA expression was raised by APS [11]. A randomized controlled trial showed that astragalus, in combination with captopril,
could significantly reduce UAE [12]. However, astragalus is an herb of warm properties and has the capacity to upset
the balance of Yin/Yang. Coptidis, is an herb of cool properties and is used to counteract the potential
harm caused by astragalus. Coptidis is used for proteinuria because not only does
it have centuries of practice-based recognition as a reliable TCM remedy for diabetes
mellitus but its potential as a treatment for DN has been established in recent evidence-based
supportive research. Liu et al. [13] evaluated the effects of compound Rhizoma Coptidis capsule (CRCC) on early DN in
rats induced by streptozotocin. Their results showed that CRCC could reduce the levels
of fasting plasma glucose (FPG), blood urea nitrogen (BUN), serum creatinine (Scr),
proteinuria, the expression of transforming growth factor-beta1 and IV-C proteins,
and alleviate pathological lesions in renal tissue of diabetic rats with nephropathy.

As a result, five Chinese medicinals are used in the present study for the treatment
of proteinuria: astragalus, coptidis, Flastem Milkvetch Seed, Gordon Euryale Seed
and Cherokee Rose Fruit (the Latin name and English name of the Chinese herbs mentioned
in the article are shown in Table 1).

One of the most obvious inconveniences of TCM is the preparation. It is very difficult
to ask patients to decoct TCM several times daily for a long time and, therefore,
this research group decided to make the formula in the form of granules (the ingredients
are shown in Table 1). The granules are processed using the following methods: first, coptidis is ethanol extracted; second, the other five herbs are decocted together and then
aqueous extracted; third, the concentrate from the previous two steps is mixed and
then spray-dried. The granule is a 10 g/package.

Objective

The aim of this clinical study is: (1) to evaluate if the efficacy of Huang Qi Elixir for proteinuria in patients with microalbuminuria is equivalent to that of
the decoction; (2) to establish the working dose of the TCM granule; and (3) to evaluate
the difference in efficacy between the TCM granule and the ARB (that is, irbesartan).

Methods/design

Design

This pilot study is a randomized, controlled, multi-center, clinical trial. The study
will be sequentially conducted as follows: enrollment after screening via inclusion
and exclusion criteria; randomization; a treatment period of 12 weeks; and assessment.
The flow chart of the study is shown in Figure 1.

6. hepatic dysfunction or a value of transaminase higher than one and a half times
the upper limit;

7. allergy to TCM/ARBs or other allergies;

8. uncontrollable urinary infection or infections which influence the measurement
of urinary protein;

9. pregnancy or lactation;

10. participation in other clinical trials within the past thirty days; and

11. anticipated survival <1 year.

Recruitment

There are six centers involved in this clinical trial and participants will be recruited
via posters at each participating center.

Randomization and intervention

The study has four arms. After enrollment, 48 participants who meet the inclusion
criteria will be randomly assigned to either: 1) granule group, 10 grams three times
daily (G10 group, n = 12); 2) granule group, 20 grams three times daily (G20 group,
n = 12); 3) the decoction group (D group, n = 12); or 4) the irbesartan group (Aprovel
group, n = 12). The treatment course is 12 weeks. The randomization sequence will
be generated with an SAS software package by the Good Clinical Practice (GCP) Center
of the Teaching Hospital of Chengdu University of TCM and will be concealed and disseminated
using opaque envelopes.

Outcome measures

Primary outcome

The primary outcome, percentage change in ACR from baseline, will be measured at week
12. ACR will be measured with a spot urine test, every four weeks; 15 to 20 ml spot
urine samples will be collected at each visit to assess albuminuria.

Secondary outcomes

The secondary outcomes include changes in GFR, Scr, HbA1c, urinalysis and FPG. All
of these will be measured every four weeks except for HbA1c, which will be measured
at week 12 only.

Statistical methods

Statistical analyses will be conducted based on an intention-to-treat population.
Participants who take at least one dose of drug and have one value on treatment comprise
the full-analysis set (FAS) and those who complete a 12-week treatment comprise the
per-protocol set (PPS). Missing data will be imputed with the use of the last-observation-carried-forward
method, whereby missing values will be replaced by the last non-missing value. Repeated
measures and multivariate analysis of variance of the general linear model will be
applied to determine the changes in GFR, Scr and FPG at each visit. Percentage reduction
from baseline in ACR and changes from baseline in HbA1c will be summarized and comparisons
will be made by using a one-way analysis of variance.

Compliance

Establishment of TCM and irbesartan after each visit will be quantified in order to
enhance medication compliance. Participants whose compliance with TCM or irbesartan
is < 80% of the total dose will be considered to have dropped out.

Adverse events

All adverse events related to TCM and irbesartan will be reported to the ethics committee
of the Teaching Hospital of Chengdu University of Traditional Chinese Medicine and
also to each participating center in written case report form. Safety will be monitored
using routine blood examination, liver and renal function, blood electrolytes and
electrocardiogram (ECG). Other potential adverse events will be clearly documented
and followed up until they are resolved or alleviated.

Sample size

Each arm will only include 12 subjects, for this is just a pilot trial aimed at exploring
some parameters for a future clinical trial with a large sample size.

Ethics

The study protocol and the written informed consent were approved by the Sichuan Regional
Ethics Review Committee on Traditional Chinese Medicine (2012KL-013). Written informed
consent will be obtained from each patient.

Discussion

Many clinical trials have been carried out to evaluate the efficacy of TCM for DN
[14,15]. Liu et al. [16] systematically reviewed randomized controlled trials evaluating the efficacy and
safety of astragalus in the treatment of DN. Their results included 33 RCTs and one
quasi-RCT involving 2,356 patients and showed that it ‘had some effects on the decrease
of the 24-hour urinary albumin excretion rate (UAER), 24-hour urinary protein, Scr,
and BUN, and also on the improvement of Ccr’ (p. 727). They concluded that it ‘has
some effect and is relatively safe in treating patients with DN’. Another study [17] evaluated the efficacy and safety of a TCM capsule, Xuezhikang, in the treatment of DN. The results of meta-analyses showed that it ‘was superior
to routine treatment in decreasing 24-h urinary protein, microalbuminuria and UAER’.
However, ‘due to a high risk of selection bias and detection bias in the included
studies, the evidence is insufficient to determine the effect of Xuezhikang’.

Obviously, it is notable that most published clinical trials which assessed the efficacy
of TCM on DN were of poor methodology, and, therefore, their results have been invalidated.
It is necessary to carry out well-designed clinical trials to provide sound evidence.
The present clinical study introduces those promising and classical herbs and formulae
into the treatment of microalbuminuria. To make administration convenient, a TCM decoction
will be processed in the form of granules. The present trial is a study of great value
for it will provide important parameters for a future randomized, placebo-controlled,
clinical trial with a large sample size.

Trial status

Patient recruitment for the trial is on-going. Data collection will continue until
the end of 2013.

Competing interests

Xiang Tu and Sen Zhong are currently applying for patents concerning Flastem Milkvetch Seed and Huang Qi Elixir formula for proteinuria/diabetic nephropathy. The other authors declare they have
no competing interests.

Authors’ contributions

XT and SZ conceived of the study. XT, FL, XFY, PF and FW designed the study. JBJ named
the formula. XT and JBJ drafted the manuscript. All authors have read and approved
the final manuscript.

Acknowledgements

The present study is supported by TCM for Prophylaxis and Treatment of Major Diseases
& Talents Training Project (2012-E-044). The funding body, Administration of TCM of
Sichuan played no role in the study. We thank all the people who have been involved
in our study.

Zhang YW, Wu CY, Cheng JT: Merit of Astragalus polysaccharide in the improvement of early diabetic nephropathy
with an effect on mRNA expressions of NF-kappaB and IkappaB in renal cortex of streptozotoxin-induced
diabetic rats.